Cathepsin proteases mediate photoreceptor cell degeneration in Drosophila

Endocytosis-mediated cell death is a form of degeneration displayed in several Drosophila mutants. This form of degeneration is displayed in several Drosophila mutant lines including flies lacking the eye-specific PLC (norpA). The cell death pathway is initiated by the stabilization of complexes between rhodopsin and arrestin which undergo massive endocytosis into the cell body. The internalized rhodopsin becomes insoluble and builds up in the late endosomal system, wherein it triggers cell death. Cathepsins are resident late endosome/lysosome proteases that have been shown to mediate apoptosis in many disease models. Therefore we sought to test the involvement of cathepsins in endocytosis-mediated retinal degeneration. Here we show that cathepsins mediate cell death in light-exposed norpA eyes. Moreover, we show that the cathepsin L-like cysteine protease, CP1, specifically mediates retinal degeneration, while the aspartyl protease, cathepsin D, does not. Furthermore, eye-specific expression of pan-cathepsin inhibitors also blocks cell death. Western blot analysis demonstrates that cathepsin L levels remain unchanged during retinal degeneration. However, whole mount immunohistochemistry performed on light-exposed retinas revealed a decrease in cathepsin L levels and a loss of rhodopsin/ CP1 colocalization, suggesting that cathepsin L translocates during the degeneration process. Lastly, we show that the retinal degeneration can be enhanced by the overexpression of cathepsin L in the sensitized norpA background. Together these data show that cathepsins play a crucial role in endocytosis-mediated retinal degeneration and are consistent with a model where rhodopsin internalization and accumulation in the endosomal/lysosomal system triggers cathepsin translocation to the cytosol.     

A Cellular Stress Model for the Differential Expression of Glial Lysosomal Cathepsins in the Aging Nervous System

Activation of the endosomal–lysosomal system and altered expression of various lysosomal hydrolases have been implicated in several senescence-dependent neurodegenerative disorders and occurs, to a lesser extent, in the course of normal brain aging. The progressive accumulation of autofluorescent, peroxidase-positive astrocytic granules represents a highly consistent biomarker of aging in the vertebrate CNS. The sulfhydryl agent cysteamine greatly accelerates the accumulation of these glial inclusionsin situand in primary brain cell cultures. We previously determined that these glial inclusions are derived from abnormal mitochondria which undergo fusion with lysosomal elements in a complex autophagic process. In the present study, we demonstrate that cysteamine suppresses cathepsin B mRNA levels and immunoreactive protein in cultured astroglia, whereas cathepsin D mRNA and protein levels are significantly augmented by CSH exposure in these cells. Moreover, cathepsin D (but not cathepsin B) exhibits robust colocalization to the red autofluorescent inclusions. Concordant with ourin vitroobservations, cathepsin B immunoreactivity is prominent in the hypothalamic ventromedial nucleus which accumulates few autofluorescent glial inclusions during aging and is relatively inapparent in the heavily granulated hypothalamic arcuate nucleus. Conversely, cathepsin D is prominent in the aging arcuate nucleus where it colocalizes to the autofluorescent inclusions and exhibits scant immunoreactivity in the adjacent ventromedial nuclear complex. In senescent astroglia, oxidative stress may down-regulate the cathepsin B gene as part of a concerted cellular stress (heat shock) response. Glial cathepsin D, on the other hand, resists stress-related inhibition and may play an important role in disposing of oxidatively modified mitochondria in the aging and degenerating nervous system.     

Experimental chloroquine myopathy: morphological and biochemical studies

Morphological and biochemical studies were performed on the soleus muscles of rats receiving a daily intraperitoneal injection of 50 mg chloroquine chloride (CQ) per kilogram of body weight. Light microscopy showed mild to moderate variations in fiber size, numerous dense membranous bodies and vacuoles. The vacuoles were ringed by material that was intensely basophilic in hematoxylin-eosin preparations, resembling rimmed vacuoles in the muscle fibers of distal myopathy. Segmental degradation or necrosis was often observed. The 3H-leucine uptake by myofibrillar and soluble sarcoplasmic fractions in CQ-treated muscles was the same as in the controls. The significant increases in lysosomal (cathepsin B & L, B and D) proteases and thiol protease inhibitor occurred in the earlier stages of CQ-induced myopathy, when hardly any autophagic vacuoles or dense bodies were observable by light microscopy. We conclude that the over-development of autophagic vacuoles and the significant increases in lysosomal protease activity in muscle tissues may be important in the development of the focal degradation and necrosis of CQ-treated muscles.     

Cathepsin D-deficient Drosophila recapitulate the key features of neuronal ceroid lipofuscinoses

Neuronal ceroid lipofuscinoses (NCLs) are a group of lysosomal storage disorders characterized pathologically by neuronal accumulation of autofluorescent storage material and neurodegeneration. An ovine NCL form is caused by a recessive point mutation in the cathepsin D gene, which encodes a lysosomal aspartyl protease. This mutation results in typical NCL pathology with neurodegeneration and characteristic neuronal storage material. We have generated a Drosophila NCL model by inactivating the conserved Drosophila cathepsin D homolog. We report here that cathepsin D mutant flies exhibit the key features of NCLs. They show progressive neuronal accumulation of autofluorescent storage inclusions, which are also positive for periodic acid Schiff and luxol fast blue stains. Ultrastructurally, the storage material is composed of membrane-bound granular electron-dense material, similar to the granular osmiophilic deposits found in the human infantile and ovine congenital NCL forms. In addition, cathepsin D mutant flies show modest age-dependent neurodegeneration. Our results suggest that the metabolic pathway leading to NCL pathology is highly conserved during evolution, and that cathepsin D mutant flies can be used to study the pathogenesis of NCLs.     

Involvement of cathepsin B in the motor neuron degeneration of amyotrophic lateral sclerosis

Abnormal proteolysis may be involved in the motor neuron degeneration of amyotrophic lateral sclerosis (ALS). Although several studies of the ubiquitin-proteasome system in ALS have been reported, the endosome-lysosome system has not been investigated in detail. To clarify the association of neurodegeneration with the endosome-lysosome system in ALS, we examined the pathological expression of cysteine proteases such as cathepsins B, H and L and an aspartate protease, cathepsin D, in the anterior horns of 15 ALS cases and 5 controls. In the ALS cases, cathepsin B immunoreactivity was preferentially decreased in the lateral parts of the anterior gray horns compared with the controls. Its immunoreactivity was increased in the cytoplasm of both shrunken and pigmented neurons but was weak in the neurons containing Bunina bodies. In addition, reactive astrocytes were also immunolabeled with cathepsin B. Cathepsin H and cathepsin L were detected in the cytoplasm of a small number of shrunken and pigmented neurons. Cathepsin D immunoreactivity was strong in the cytoplasm of all motor neurons. The immunoreactivity of cathepsins H, L and D was not significantly different between control and ALS cases. Western blot analysis showed that the 25-kDa activated form of cathepsin B was down-regulated in ALS. Our results suggest that cathepsin B is involved in the motor neuron degeneration in ALS.     

Cathepsin B contributes to traumatic brain injury‐induced cell death through a mitochondria‐mediated apoptotic pathway

It has been reported that lysosomal proteases play important roles in ischemic and excitotoxic neuronal cell death. We have previously reported that cathepsin B expression increased remarkably after traumatic brain injury (TBI). The present study sought to investigate the effects of a selective cathepsin B inhibitor (CBI) [N‐L‐3‐trans‐prolcarbamoyloxirane‐2‐carbonyl)‐L‐isoleucyl‐L‐proline] on cell death and behavioral deficits in our model. We examined the levels of cathepsin B enzymatic activity and its expression by double labelling damaged cells in the brain slice with propidium iodide (PI) and anticathepsin B. The results showed an elevated enzymatic activity associated with TBI‐induced increase in a mature form of cathepsin B, suggesting that cathepsin B may play a role in TBI‐induced cell injury. PI was found to label cells positive for the neuronal‐specific nuclear marker NeuN, whereas fewer GFAP‐positive cells were labelled by PI, suggesting that neurons are more sensitive to cell death induced by TBI. Additionally, we found that pretreatment with CBI remarkably attenuated TBI‐induced cell death, lesion volume, and motor and cognitive dysfunction. To analyze the mechanism of action of cathepsin B in the cell death signaling pathway, we assessed DNA fragmentation by electrophoresis, Bcl‐2/Bax protein expression levels, Bid cleavage, cytochrome c release, and caspase‐3 activation. The results imply that cathepsin B contributes to TBI‐induced cell death through the present programmed cell necrosis and mitochondria‐mediated apoptotic pathways. © 2010 Wiley‐Liss, Inc.     

Lysosomal Cathepsin Protease Gene Expression Profiles in the Human Brain During Normal Development

Cathepsin protease genes are necessary for protein homeostasis in normal brain development and function. The diversity of the 15 cathepsin protease activities raises the question of what are the human brain expression profiles of the cathepsin genes during development from prenatal and infancy to childhood, adolescence, and young adult stages. This study, therefore, evaluated the cathepsin gene expression profiles in 16 human brain regions during development by quantitative RNA-sequencing data obtained from the Allen Brain Atlas resource. Total expression of all cathepsin genes was the lowest at the early prenatal stage which became increased at the infancy stage. During infancy to young adult phases, total gene expression was similar. Interestingly, the rank ordering of gene expression among the cathepsins was similar throughout the brain at the age periods examined, showing (a) high expression of cathepsins B, D, and F; (b) moderate expression of cathepsins A, L, and Z; (c) low expression of cathepsins C, H, K, O, S, and V; and (d) very low expression of cathepsins E, G, and W. Results show that the human brain utilizes well-defined, balanced patterns of cathepsin gene expression throughout the different stages of human brain development. Knowledge gained by this study of the gene expression profiles of lysosomal cathepsin proteases among human brain regions during normal development is important for advancing future investigations of how these cathepsins are dysregulated in lysosomal-related brain disorders that affect infants, children, adolescents, and young adults.     

Cathepsin B and L are involved in degradation of prions in GT1-1 neuronal cells

In scrapie-infected cells, the abnormal isoform of the prion protein, PrP(Sc), accumulates in endosomes/lysosomes. In this study, the involvement of two lysosomal proteases, cathepsin B and L, in cellular processing of PrP(Sc) was analyzed in immortalized neuronal gonadotropin-releasing hormone cells (GT1-1) infected with scrapie. Treatment with inhibitors of either cathepsin B or L resulted in accumulation of PrP(Sc). Such an increased accumulation also occurred when the activities of both cathepsins were inhibited using RNA interference. We conclude that cathepsin B and L are involved in the degradation of PrP(Sc) in scrapie-infected GT1-1 cells and that they can compensate for each other's functions. This study shows that specific proteases, abundantly present in neurons, have the capacity to degrade PrP(Sc).     

Enhanced cystatin C and lysosomal protease expression following 6-hydroxydopamine exposure

6-Hydroxydopamine (6-OHDA) is a selective neurotoxin used to induce apoptosis in catecholamine-containing neurons. Although biochemical products and reactive oxygen species (ROS) of 6-OHDA have been well documented, the activation of cellular pathways following exposure are not well understood. Apoptosis in PC12 (Pheochromocytoma) cells was induced by 6-OHDA in a dose (10-150 microM) and time-dependent (24-72 h) manner compared to experimental controls (no treatment). PC 12 cells exposed to 50 microM 6-OHDA demonstrated the involvement of caspase 3 and lysosomal protease alterations. Following 6-OHDA exposure, the caspase 3-like inhibitor Ac-DEVD-CHO significantly decreased 6-OHDA induced cell death. In addition, alterations in expression of the lysosomal cysteine and aspartic proteases, cathepsin B (CB) and cathepsin D (CD) and the endogenous cysteine protease inhibitor cystatin C were observed utilizing immunocytochemical analysis at 24, 48, and 72 h following 6-OHDA exposure. Furthermore, CB and CD and cystatin C immuno-like reactivity was more pronounced in TUNEL positive cells. Moreover, Western blot analysis confirmed a significant increase in protein expression for CB and CD at 72 h and a temporal and concentration dependent increase in cystatin C in response to 6-OHDA. Cells treated with pepstatin A, an inhibitor for CD, showed a significant decrease in cell death, however, CA-074ME, a specific inhibitor for CB, failed to protect cells from 6-OHDA induced cell death. Thus, these results suggest that apoptosis induced by 6-OHDA exposure is mediated in part through caspase 3 activation and lysosomal protease CD.     

P38 MAPK is involved in enhanced NMDA receptor-dependent excitotoxicity in YAC transgenic mouse model of Huntington disease

Huntington disease (HD) is a dominantly inherited neurodegenerative disease caused by a polyglutamine (polyQ) expansion in the protein huntingtin (htt). Previous studies have shown enhanced N-methyl-d-aspartate (NMDA)-induced excitotoxicity in neuronal models of HD, mediated in part by increased NMDA receptor (NMDAR) GluN2B subunit binding with the postsynaptic density protein-95 (PSD-95). In cultured hippocampal neurons, the NMDAR-activated p38 Mitogen-activated Protein Kinase (MAPK) death pathway is disrupted by a peptide (Tat-NR2B9c) that uncouples GluN2B from PSD-95, whereas NMDAR-mediated activation of c-Jun N-terminal Kinase (JNK) MAPK is PSD-95-independent. To investigate the mechanism by which Tat-NR2B9c protects striatal medium spiny neurons (MSNs) from mutant htt (mhtt)-enhanced NMDAR toxicity, we compared striatal tissue and cultured MSNs from presymptomatic yeast artificial chromosome (YAC) mice expressing htt with 128 polyQ (YAC128) to those from YAC18 and/or WT mice as controls. Similar to the previously published shift of GluN2B-containing NMDARs to extrasynaptic sites, we found increased PSD-95 localization as well as elevated PSD-95-GluN2B interactions in the striatal non-PSD (extrasynaptic) fraction from YAC128 mice. Notably, basal levels of both activated p38 and JNK MAPKs were elevated in the YAC128 striatum. NMDA stimulation of acute slices increased activation of p38 and JNK in WT and YAC128 striatum, but Tat-NR2B9c pretreatment reduced only the p38 activation in YAC128. In cultured MSNs, p38 MAPK inhibition reduced YAC128 NMDAR-mediated cell death to WT levels, and occluded the Tat-NR2B9c peptide protective effect; in contrast, inhibition of JNK had a similar protective effect in cultured MSNs from both WT and YAC128 mice. Our results suggest that altered activation of p38 MAPK contributes to mhtt enhancement of GluN2B/PSD-95 toxic signaling.     

Immunolocalization of cathepsins B,H and L in skeletal muscle of X-linked muscular dystrophy (mdx) mouse

Summary The amounts of non-collagen proteins (muscle structural proteins) and the activity of creatine kinase were significantly decreased in muscles of 28-day-old mdx mice. The activities of lysosomal thiol proteases such as cathepsins B and L were increased in muscles of mdx mice at as early as 10 days of age. Endogenous thiol proteinase inhibitor and various lysosomal hydrolases also showed increased activities. The localization of cathepsins B, H and L, and endogenous thiol proteinase inhibitor was investigated using the respective specific antibodies. While only invading macrophages were stained strongly with anticathepsin B and H, and anti-thiol proteinase inhibitor antibodies, cathepsin L was localized in muscle cells as well as in invading macrophages. Cathepsin L in muscle cells itself may initially degrade muscle structural proteins, before lysosomal thiol proteases, mainly derived from macrophages, degrade them in skeletal muscles of mdx mice.     

Elevations of cathepsin B and cathepsin L activities in forelimb and hind limb muscles of genetically dystrophic mice

The combined activities of cathepsin B and cathepsin L were studied in the forelimb and hind limb muscles of dystrophic mice. The activities of these proteases in the forelimb and hind limb muscles of young and adult dystrophic mice were significantly higher than those in normal mice. However, clinical involvement of dystrophy appeared in the hind limbs but not in the forelimbs. We therefore suggest that the increase in protease activity begins at a very early age and that the clinical involvement is not linked with the increase in cathepsin B and L.     

Huntingtin is degraded to small fragments by calpain after ischemic injury

The gene defect in Huntington's disease (HD) causes a polyglutamine expansion in the N-terminal region of huntingtin (N-htt). In vitro studies suggest that mutant N-htt fragments can aggregate and cause cell death in HD. The physiological and pathological conditions that affect htt proteolysis in the brain are unclear. We examined htt expression by Western blot in the rat brain after transient ischemic injury, which causes striatal neurodegeneration similar to that seen in HD and activates proteases including calcium-dependent calpains. Focal brain ischemia reduced levels of full-length htt in the infarcted cortex and striatum and increased expression of a 55-kDa N-htt fragment that was also produced by treating control brain extracts with calpain. N-htt fragments between 65 and 80 kDa also rose after injury, but these fragments were not as long-lived as the 55-kDa N-htt fragment. The results suggest that after ischemic injury full-length htt is degraded in degenerating neurons and an N-htt fragment accumulates.     

Alterations of the posttranslational processing of a lysosomal enzyme in C6 glioma cells

Cathepsin D was assessed in C6 glioma cells grown in medium with an intermediate- or low-percent composition of serum. The amount, form, and subcellular location of cathepsin D differed after treatment with cyanate or monensin in cells grown in a low-serum, growth-factor-supplemented medium. Immunoblotting showed that cathepsin D in the lysosomal fraction of the C6 cell line had a molecular weight (Mr) of 42 kD, whereas that in the microsomal fraction had Mr's of 42, 47, and 78 kD. After treatment for 1 to 16 hr with 4 mmol/L cyanate and subcellular fractionation, the molecular weight of lysosomal cathepsin D was the same in treated and untreated cells, but more enzyme was found in lysosomes of treated cells at 8 and 16 hr. In the microsomal fraction, the amounts of both the 42 and 47 kD forms were increased after 1 to 16 hr of treatment. When exposed to 20 mmol/L cyanate, C6 cells remained viable, but compared with untreated cells, they showed 25% less lysosomal cathepsin D, with increased amounts found in the microsomal fraction. The 78 kD protein detected by immunoblotting was present in both the lysosomal and microsomal fractions but was predominant in the latter. The apparent molecular weight of this protein was the same after cyanate but differed with monensin, where Mr's of 39, 42, and 73 kD were found. Monensin-treated cells had less lysosomal cathepsin D and relatively more microsomal enzyme. The differing molecular weights of cathepsin D from cyanate- and monensin-treated cells suggest that their inhibitions occur at different processing loci in distal elements of the Golgi stacks. The differences in the pI of cathepsin D and the number of its forms from cyanate- and monensin-treated cells are also consistent with interference in the late stages of glycoprotein maturation. In this paper we show that the amount, molecular form, and consequent intracellular location of cathepsin D in cells of the C6 line can be affected by agents that selectively disrupt stages in Golgi-related protein modification and transport.     

A comparison of cathepsin B processing and distribution during neuronal death in rats following global ischemia or decapitation necrosis

The objective of this study was to examine the possible role of the cysteine protease cathepsin B (E.C. 3.4.22.1) in the delayed neuronal death in rats subjected to the two-vessel occlusion model of global ischemia. Immunohistochemistry of the hippocampus showed an alteration in the distribution of cathepsin B in CA1 neurons from a lysosomal pattern to a more intense label redistributed into the cytoplasm. This change was not detected until the neurons had become morphologically altered with obvious shrinkage of the cytoplasmic region. Western blotting and enzyme activity measurements of subcellular fractions, including lysosomes and a cell soluble fraction, demonstrated that there was an overall decrease in cathepsin B activity at this time but an increase in the proenzyme form, particularly in the soluble fraction. This was found to be completely different from the marked loss of all forms of cathepsin B in necrotic neurons following decapitation. © 1997 Elsevier Science B.V. All rights reserved.     

Oxidative stress-induced phosphorylation, degradation and aggregation of alpha-synuclein are linked to upregulated CK2 and cathepsin D

Intracellular accumulation of alpha-synuclein (alpha-Syn) as filamentous aggregates is a pathological feature shared by Parkinson's disease, dementia with Lewy bodies and multiple system atrophy, referred to as synucleinopathies. To understand the mechanisms underlying alpha-Syn aggregation, we established a tetracycline-off inducible transfectant (3D5) of neuronal lineage overexpressing human wild-type alpha-Syn. Alpha-Syn aggregation was initiated by exposure of 3D5 cells to FeCl2. The exposure led to formation of alpha-Syn inclusions and oligomers of 34, 54, 68 kDa and higher molecular weights. The oligomers displayed immunoreactivity with antibodies to the amino-, but not to the carboxyl (C)-, terminus of alpha-Syn, indicating that C-terminally truncated alpha-Syn is a major component of oligomers. FeCl2 exposure also promoted accumulation of S129 phosphorylated monomeric alpha-Syn (P alpha-Syn) and casein kinase 2 (CK2); however, G-protein-coupled receptor kinase 2 was reduced. Treatment of FeCl2-exposed cells with CK2 inhibitors (DRB or TBB) led to decreased formation of alpha-Syn inclusions, oligomers and P alpha-Syn. FeCl2 exposure also enhanced the activity/level of cathepsin D. Treatment of the FeCl2-exposed cells with pepstatin A or NH4Cl led to reduced formation of oligomers/inclusions as well as of approximately 10 and 12 kDa truncated alpha-Syn. Our results indicate that alpha-Syn phosphorylation caused by FeCl2 is due to CK2 upregulation, and that lysosomal proteases may have a role in producing truncated alpha-Syn for oligomer assembly.     

Cathepsin D displays in vitro β-secretase-like specificity

The formation of Aβ and Aβ-containing fragments is likely a key event in the process of neural degeneration in Alzheimer's disease. The N-terminal residue (Asp-1) of Aβ and its C-terminally extended sequences is liberated from the β-amyloid precursor protein (βAPP) by β-secretase(s). This activity appears highly increased by the presence (N-terminally to Asp-1) of a double-mutation (KM→NL) found in several Swedish families affected by early onset Alzheimer's disease. By means of synthetic peptides encompassing the ‘normal' (N peptide) and mutated (ΔNL peptide) sequences targeted by β-secretase(s), we have detected a human brain protease displaying preferred efficiency for the ΔNL peptide than for the non-mutated analog. This activity is sensitive to pepstatin, maximally active at acidic pH and hydrolyses the two peptides at the expected M/D or L/D cleavage sites. Such acidic activity is also detected in rat brain, PC12 cells and primary cultured astrocytes. The pepstatin sensitivity and pH maximum of the brain activity that appeared reminiscent of those displayed by the acidic protease cathepsin D led us to examine this enzyme as a putative β-secretase-like candidate. Purified cathepsin D displays higher catalytic parameters for the ΔNL peptide than for the non-mutated peptide, cleaves these two substrates at the expected M/D or L/D sites, and is maximally active at acidic pH. However, cathepsin D does not cleave peptides bearing mutations that were previously shown to drastically lower or fully block Aβ secretion by transfected cells. Furthermore, cathepsin D hydrolyses recombinant baculoviral ΔNLβAPP₇₅₁ at a 6-fold higher rate than βAPP₇₅₁ and gives rise to a 12-kDa C-terminal product that is recognized by antibodies fully specific of the N-terminus of Aβ. Altogether, our study indicates that cathepsin D displays several in vitro β-secretase-like properties that suggests that this protease could fulfill such a role, at least in the Swedish genetic form of Alzheimer's disease. © 1997 Elsevier Science B.V. All rigths reserved.     

Huntingtin and its role in neuronal degeneration.

Huntington's disease results from a polyglutamine expansion in the N-terminal region of huntingtin (htt). This abnormality causes protein aggregation and leads to neurotoxicity. Despite its widespread expression in the brain and body, mutant htt causes selective neurodegeneration in Huntington's disease patient brains. However, Huntington's disease mouse models expressing mutant htt do not have obvious neurodegeneration despite significant neurological symptoms. Most Huntington's disease mouse models display the accumulation of toxic N-terminal mutant htt fragments in both the nucleus and neuronal processes, suggesting that these subcellular sites are hotspots for the early neuropathology of Huntington's disease. Intranuclear htt affects gene expression and may cause neuronal dysfunction. Mutant htt in neuronal processes affects axonal transport and induces degeneration, and these effects may be more relevant to the selective neurodegeneration in Huntington's disease. Growing evidence has also suggested that mutant htt mediates multiple pathological pathways. This review discusses the early pathological changes identified in Huntington's disease cellular and animal models. These changes may be the causes of neurode-generation.     

Association between cathepsin D polymorphism and Alzheimer's disease in a Chinese Han population

Cathepsin D (CTSD) is an intracellular aspartyl protease, which is active in the endosomal/lysosomal system. CTSD may play a role in Alzheimer's disease (AD) through cleaving the amyloid precursor protein into beta-amyloid peptide and degrading tau protein into fragments. A functional polymorphism in exon 2 of the cathepsin D gene (C-->T, Ala224Val) has recently been reported to increase the risk for AD in some of the Caucasian populations, with a significant overrepresentation of the T allele, but these reports have not been universally duplicated. We performed an association study between CTSD polymorphism and AD in 156 sporadic AD patients and 183 controls of Chinese Han ethnicity. Our data revealed that the distribution of CTSD genotypes and alleles was similar in patients and controls. No direct association was found between CTSD polymorphism and AD risk. There might be a weak synergistic interaction between CTSD T and APOEepsilon4 allele in increasing the risk for developing AD.     

Characterization of huntingtin pathologic fragments in human Huntington disease, transgenic mice, and cell models

Huntington disease (HD) is caused by the expansion of a glutamine (Q) repeat near the N terminus of huntingtin (htt), resulting in altered conformation of the mutant protein to produce, most prominently in brain neurons, nuclear and cytoplasmic inclusion pathology. The inclusions and associated diffuse accumulation of mutant htt in nuclei are composed of N-terminal fragments of mutant protein. Here, we used a panel of peptide antibodies to characterize the htt protein pathologies in brain tissues from human HD, and a transgenic mouse model created by expressing the first 171 amino acids of human htt with 82Q (htt-N171-82Q). In tissues from both sources, htt pathologic features in nuclei were detected by antibodies to htt peptides 1-17 and 81-90 but not 115-129 (wild-type huntingtin numbering with 23 repeats). Human HEK 293 cells transfected with expression vectors that encode either the N-terminal 233 amino acids of human htt (htt-N233-82Q) or htt-N171-18Q accumulated smaller N-terminal fragments with antibody-binding characteristics identical to those of pathologic peptides. We conclude that the mutant htt peptides that accumulate in pathologic structures of human HD and httN171-82Q in mice are produced by similar, yet to be defined, proteolytic events in a region of the protein near or within amino acids 90-115.